Rashwan Nour, Scott Marilyn, Prichard Roger
Institute of Parasitology, Macdonald College, McGill University, Ste Anne de Bellevue, Quebec, Canada.
PLoS Negl Trop Dis. 2017 Jan 12;11(1):e0005205. doi: 10.1371/journal.pntd.0005205. eCollection 2017 Jan.
The benzimidazole (BZ) anthelmintics, albendazole (ABZ) and mebendazole (MBZ) are the most common drugs used for treatment of soil-transmitted helminths (STHs). Their intensive use increases the possibility that BZ resistance may develop. In veterinary nematodes, BZ resistance is caused by a single nucleotide polymorphism (SNP) in the β-tubulin isotype 1 gene at codon position 200, 167 or 198, and these SNPs have also been correlated with poor response of human Trichuris trichiura to BZ treatment. It is important to be able to investigate the presence of resistance-associated SNPs in STHs before resistance becomes clinically established.
The objective of this study was to develop new genotyping assays to screen for the presence of β-tubulin SNPs in T. trichiura and Ascaris lumbricoides. Rapid, simple and accurate genotyping assays were developed based on the SmartAmp2 method. Primer sets were optimized and selected to distinguish the SNP-variant genotypes. After initial optimization on control plasmids, the feasibility of the assay was assessed in field samples from Haiti and Panama. Finally, spiked fecal samples were assessed to determine the tolerance of Aac polymerase to fecal inhibitors.
Rapid SNP genotyping assays were developed to target β-tubulin polymorphisms in T. trichiura and A. lumbricoides. The assays showed high sensitivity and specificity in field samples and also demonstrated high tolerance to PCR inhibitors in fecal samples.
These assays proved to be robust and efficient with the potential to be used as field tools for monitoring SNPs that could be associated with BZ resistance. However, further work is needed to validate the assays on large numbers of field samples before and after treatment.
苯并咪唑（BZ）类驱虫药阿苯达唑（ABZ）和甲苯达唑（MBZ）是治疗土源性蠕虫（STH）最常用的药物。它们的大量使用增加了产生BZ耐药性的可能性。在兽用线虫中，BZ耐药性是由β-微管蛋白1型基因密码子位置200、167或198处的单核苷酸多态性（SNP）引起的，这些SNP也与人类毛首鞭形线虫对BZ治疗反应不佳有关。在耐药性在临床上确立之前，能够调查STH中耐药相关SNP的存在情况非常重要。
本研究的目的是开发新的基因分型检测方法，以筛查毛首鞭形线虫和蛔虫中β-微管蛋白SNP的存在情况。基于SmartAmp2方法开发了快速、简单且准确的基因分型检测方法。对引物组进行了优化和筛选，以区分SNP变异基因型。在对照质粒上进行初步优化后，在来自海地和巴拿马的现场样本中评估了该检测方法的可行性。最后，对加标的粪便样本进行评估，以确定Aac聚合酶对粪便抑制剂的耐受性。
开发了针对毛首鞭形线虫和蛔虫中β-微管蛋白多态性的快速SNP基因分型检测方法。这些检测方法在现场样本中显示出高灵敏度和特异性，并且对粪便样本中的PCR抑制剂也表现出高耐受性。
这些检测方法被证明是强大且高效的，有潜力用作监测可能与BZ耐药性相关的SNP的现场工具。然而，在治疗前后对大量现场样本进行检测方法的验证之前，还需要进一步的工作。
